A new approach methodology using kinetically-derived maximum dose levels in risk assessment - A case study with afidopyropen.

We present a case study for afidopyropen (AF; insecticide) to characterize chronic dietary human health risk using a Risk 21-based approach. Our objective is to use a well-tested pesticidal active ingredient (AF) to show how a new approach methodology (NAM), using the kinetically-derived maximum dose (KMD) and with far less animal testing, can reliably identify a health-protective point of departure (PoD) for chronic dietary human health risk assessments (HHRA). Chronic dietary HHRA involves evaluation of both hazard and exposure information to characterize risk. Although both are important, emphasis has been placed on a checklist of required toxicological studies for hazard characterization, with human exposure information only considered after evaluation of hazard data. Most required studies are not used to define the human endpoint for HHRA. The information presented demonstrates a NAM that uses the KMD determined by saturation of a metabolic pathway, which can be used as an alternative POD. In these cases, the full toxicological database may not need to be generated. Demonstration that the compound is not genotoxic and that the KMD is protective of adverse effects in 90-day oral rat and reproductive/developmental studies is sufficient to support the use of the KMD as an alternative POD.

Rapid equilibrium dialysis, ultrafiltration or ultracentrifugation? Evaluation of methods to quantify the unbound fraction of substances in plasma.

In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (fu) is arguably regarded the effective concentration at the target site. Pharmacology and toxicology, increasingly use in vitro models. The translation of in vitro concentrations to in vivo doses can be supported by toxicokinetic modelling, e.g. physiologically based toxicokinetic models (PBTK). PPB of a test substance is an input parameter for PBTK. We compared three methods to quantify fu: rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC) using twelve substances covering a wide range of Log Pow (-0.1 to 6.8) and molecular weights (151 and 531 g/mol): Acetaminophen, Bisphenol A, Caffeine, Colchicine, Fenarimol, Flutamide, Genistein, Ketoconazole, α-Methyltestosterone, Tamoxifen, Trenbolone and Warfarin. After RED and UF separation, three polar substances (Log Pow < 2) were largely unbound (fu > 70%), while more lipophilic substances were largely bound (fu < 33%). Compared to RED or UF, UC resulted in a generally higher fu of lipophilic substances. fu obtained after RED and UF were more consistent with published data. For half of the substances, UC resulted in fu higher than the reference data. UF, RED and both UF and UC, resulted in lower fu of Flutamide, Ketoconazole and Colchicine, respectively. For fu quantifications, the separation method should be selected according to the test substance's properties. Based on our data, RED is suitable for a broader range of substances while UC and UF are suitable for polar substances.

Toward Realistic Dosimetry In Vitro: Determining Effective Concentrations of Test Substances in Cell Culture and Their Prediction by an In Silico Mass Balance Model.

Nominal concentrations (CNom) in cell culture media are routinely used to define concentration-effect relationships in the in vitro toxicology. The actual concentration in the medium (CMedium) can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log KOW -0.07-6.84), in the culture medium (CMedium) and cells (CCell) after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished in silico mass balance model that combined relevant equations from similar models published by others. The total CMedium for all chemicals except tamoxifen (TAM) were similar to the CNom. This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) CMedium for the low/no protein binding chemicals were similar to the CNom, whereas values of all moderately to highly protein-bound chemicals were less than 30% of the CNom. Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the CCell was the same as the CNom. The CCell for all other chemicals tended to increase over time and were all 2- to 274-fold higher than CNom. Measurements of CCytosol, using a digitonin method to release cytosol, compared well with CCell (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total CMedium within 30% of the measured values for 11 chemicals. The free CMedium of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of CCell values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between CNom and total and free CMedium and CCell, which were also relatively well predicted by the mass balance model.

Predictive performance of next generation human physiologically based kinetic (PBK) models based on in vitro and in silico input data.

The goal of the present study was to assess the predictive performance of a generic human physiologically based kinetic (PBK) model based on in vitro and in silico input data and the effect of using different input approaches for chemical parameterization on those predictions. For this purpose, a dataset was created of 38,772 Cmax predictions for 44 compounds by applying different combinations of in vitro and in silico approaches for chemical parameterization, and these predicted Cmax values were compared to reported in vivo data. Best results were achieved when the hepatic clearance was parameterized based on in vitro (i.e., hepatocytes or liver S9) measured intrinsic clearance values, the method of Rodgers and Rowland for calculating tissue:plasma partition coefficients, and the method of Lobell and Sivarajah for calculating the fraction unbound in plasma. With these parameters, the median Cmax values of 34 out of the 44 compounds were predicted within 5-fold of the observed Cmax, and the Cmax values of 19 compounds were predicted within 2-fold. The median Cmax values of 10 compounds were more than 5-fold overestimated. Underestimations (> 5-fold) did not occur. A comparison of the current generic PBK model structure with chemical-specific PBK models available in literature was made to identify possible kinetic processes not included in the generic PBK model that might explain the overestimations. Overall, the results provide crucial insights into the predictive performance of PBK models based on in vitro and in silico input and the influence of different input approaches on the model predictions.

Predictive Performance of Next Generation Physiologically Based Kinetic (PBK) Model Predictions in Rats Based on In Vitro and In Silico Input Data.

The goal of the present study was to assess the predictive performance of a minimal generic rat physiologically based kinetic (PBK) model based on in vitro and in silico input data to predict peak plasma concentrations (Cmax) upon single oral dosing. To this purpose, a dataset was generated of 3960 Cmax predictions for 44 compounds, applying different combinations of in vitro and in silico approaches for chemical parameterization, and comparison of the predictions to reported in vivo data. Best performance was obtained when (1) the hepatic clearance was parameterized based on in vitro measured intrinsic clearance values, (2) the method of Rodgers and Rowland for calculating partition coefficients, and (3) in silico calculated fraction unbound plasma and Papp values (the latter especially for very lipophilic compounds). Based on these input data, the median Cmax of 32 compounds could be predicted within 10-fold of the observed Cmax, with 22 out of these 32 compounds being predicted within 5-fold, and 8 compounds within 2-fold. Overestimations of more than 10-fold were observed for 12 compounds, whereas no underestimations of more than 10-fold occurred. Median Cmax predictions were frequently found to be within 10-fold of the observed Cmax when the scaled unbound hepatic intrinsic clearance (Clint,u) was either higher than 20 l/h or lower than 1 l/h. Similar findings were obtained with a test set of 5 in-house BASF compounds. Overall, this study provides relevant insights in the predictive performance of a minimal PBK model based on in vitro and in silico input data.

Enigmatic mechanism of the N-vinylpyrrolidone hepatocarcinogenicity in the rat.

N-vinyl pyrrolidone (NVP) is produced up to several thousand tons per year as starting material for the production of polymers to be used in pharmaceutics, cosmetics and food technology. Upon inhalation NVP was carcinogenic in the rat, liver tumor formation is starting already at the rather low concentration of 5 ppm. Hence, differentiation whether NVP is a genotoxic carcinogen (presumed to generally have no dose threshold for the carcinogenic activity) or a non-genotoxic carcinogen (with a potentially definable threshold) is highly important. In the present study, therefore, the existing genotoxicity investigations on NVP (all showing consistently negative results) were extended and complemented with investigations on possible alternative mechanisms, which also all proved negative. All tests were performed in the same species (rat) using the same route of exposure (inhalation) and the same doses of NVP (5, 10 and 20 ppm) as had been used in the positive carcinogenicity test. Specifically, the tests included an ex vivo Comet assay (so far not available) and an ex vivo micronucleus test (in contrast to the already available micronucleus test in mice here in the same species and by the same route of application as in the bioassay which had shown the carcinogenicity), tests on oxidative stress (non-protein-bound sulfhydryls and glutathione recycling test), mechanisms mediated by hepatic receptors, the activation of which had been shown earlier to lead to carcinogenicity in some instances (Ah receptor, CAR, PXR, PPARα). No indications were obtained for any of the investigated mechanisms to be responsible for or to contribute to the observed carcinogenicity of NVP. The most important of these exclusions is genotoxicity. Thus, NVP can rightfully be regarded and treated as a non-genotoxic carcinogen and threshold approaches to the assessment of this chemical are supported. However, the mechanism underlying the carcinogenicity of NVP in rats remains unclear.

Afidopyropen: Challenges and impact of a toxicokinetic study designed to identify a point of inflection from dose-proportionality.

Afidopyropen is an insecticide that acts as a transient receptor potential vanilloid subtype (TRPV) channel modulator in chordotonal organs of target insects and has been assessed for a wide range of toxicity endpoints including chronic toxicity and carcinogenicity in rats and mice. The current study evaluates the toxicokinetic properties of afidopyropen and its plasma metabolites in rats at dose levels where the pharmacokinetics (PK) are linear and nonlinear in an attempt to identify a point of inflection. Based on the results of this study and depending on the analysis method used, the kinetically derived maximum dose (KMD) is estimated to be between 2.5 and 12.5 mg/kg bw/d with linearity observed at doses below 2.5 mg/kg bw/d. A defined point of inflection could not be determined. These data demonstrate that consideration of PK is critical for improving the dose-selection in toxicity studies as well as to enhance human relevance of the interpretation of animal toxicity studies. The study also demonstrates the technical difficulty in obtaining a defined point of inflection from in vivo PK data.

Metabolism and plasma protein binding of 16 straight- and branched-chain parabens in in vitro liver and skin models.

Parabens are alkyl esters of 4-hydroxybenzoic acid (4-HBA), with short-chain parabens used as antimicrobials in cosmetics. We investigated the impact of chain structure on skin and liver metabolism. Incubations with primary human hepatocytes and human liver S9 indicated that methyl-, ethyl-, propyl- and butylparaben were rapidly metabolized to similar metabolites, including 4-HBA plus the corresponding alcohols. Liver and EpiSkin™ S9 were used to investigate the metabolism of 16 short and long straight- and branched-chain parabens. The rate of hydrolysis generally decreased with increasing chain length in liver S9, whereas the reverse was true for EpiSkin™ S9. Chain length also correlated with the number of metabolites, with more oxidized metabolites detected from longer chain parabens. The identity of the alcohol group impacted metabolism the most, in terms of the rate of metabolism and the contribution of cofactors. The majority of parabens (13/16) exhibited high plasma protein binding (PPB) (>90%); whereas, 4-HBA PPB was 38%. PPB was related to the LogP of the parabens. In conclusion, the major and common paraben metabolite in PHH, liver S9 and EpiSkin™ S9 was 4-HBA. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific (liver, skin) and was influenced by the chain length (and hence LogP), structural isomeric form (straight vs branched), and/or the identity of the alkyl group. SHORT ABSTRACT: We investigated how the chain structure of parabens affects their metabolism by liver and EpiSkin™ S9. The major and common metabolite in primary human hepatocytes, liver S9 and EpiSkin™ S9 was 4-HBA plus the corresponding alcohols. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific and influenced by the chain length, structural isomeric form (straight vs branched), and/or the identity of the alkyl group. Most parabens exhibited high PPB (>90%), whereas the PPB of 4-HBA was 38%.

Novel testing strategy for prediction of rat biliary excretion of intravenously administered estradiol-17ß glucuronide.

The aim of the present study was to develop a generic rat physiologically based kinetic (PBK) model that includes a novel testing strategy where active biliary excretion is incorporated using estradiol-17ß glucuronide (E217ßG) as the model substance. A major challenge was the definition of the scaling factor for the in vitro to in vivo conversion of the PBK-model parameter Vmax. In vitro values for the Vmax and Km for transport of E217ßG were found in the literature in four different studies based on experiments with primary rat hepatocytes. The required scaling factor was defined based on fitting the PBK model-based predicted values to reported experimental data on E217ßG blood levels and cumulative biliary E217ßG excretion. This resulted in a scaling factor of 129 mg protein/g liver. With this scaling factor the PBK model predicted the in vivo data for blood and cumulative biliary E217ßG levels with on average of less than 1.8-fold deviation. The study provides a proof of principle on how biliary excretion can be included in a generic PBK model using primary hepatocytes to define the kinetic parameters that describe the biliary excretion.

In vitro dermal absorption of metiram using human skin preparations.

Metiram is a polymeric plant protection fungicidal product. Since farm workers can potentially be exposed to the used solo-formulation, polyram, its dermal penetration is important for the assessment of its safety. Previous dermal penetration studies indicated a low penetration of metiram (≤ 1% of the applied dose), when applied in polyram or in the almost identical technical concentrate, metiram TK. Here, we present an in vitro human skin absorption study conducted according to OECD guideline 428. In this study, synthesized polymeric 14C-radiolabelled metiram in polymeric "unlabeled" solo-formulation, polyram, was used. Single doses of radioactive metiram were applied to human skin preparations in vitro (4 donors per dose group, 2 replicates each) for 8 h, under semi-occluded conditions in Franz-like diffusion cells, using a flow-through diffusion system. Under these test conditions, dermal absorption of the low dose, which represents spray dilution concentrations, was 0.34 ± 0.48% of applied dose; and dermal absorption of the high dose (formulation concentrate) was negligible (<0.0015 % of the applied dose). These results confirm the low dermal absorption of polymeric metiram and indicate that slight differences in applied formulations have minimal impact on its penetration properties.

Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

Use of toxicokinetic data for afidopyropen to determine the dose levels in developmental toxicity studies.

Afidopyropen is an insecticide that acts as a TRPV channel modulator in chordotonal organs of target insects and has been assessed for a wide range of toxicity endpoints including developmental toxicity in rats and rabbits. The GLP developmental toxicity study in rabbits did not produce evidence of maternal or fetal toxicity at the highest dose tested (32 mg/kg/day) but pharmacokinetics (PK) in pregnant rabbits in this study exhibited onset of PK nonlinearity from 5 mg/kg/day on, as measured by plasma Cmax and AUC. The NOAEL (32 mg/kg/day) is 9000X higher than maximum expected human dietary exposures to afidopyropen; the dose range where nonlinear PK were observed (5-15 mg/kg/day) is 1400-4200X higher. As nonlinearity occurred between 5 and 15 mg/kg/day, 32 mg/kg/day is concluded to be a sufficiently high dose (kinetically derived maximum dose) for a prenatal developmental toxicity study. As recognized by regulatory dose-selection guidance, onset of saturated PK is evidence of excessive biological stress to test animals rendering any effects at such doses of questionable relevance for human risk assessment. These data demonstrate that consideration of PK is critical for improving the dose-selection in developmental toxicity studies to enhance human relevance of animal toxicity studies.

Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol.

OECD test guideline 428 compliant protocol using human skin was used to test the penetration of 56 cosmetic-relevant chemicals. The penetration of finite doses (10 µL/cm2 ) of chemicals was measured over 24 hours. The dermal delivery (DD) (amount in the epidermis, dermis and receptor fluid [RF]) ranged between 0.03 ± 0.02 and 72.61 ± 8.89 µg/cm2 . The DD of seven chemicals was comparable with in vivo values. The DD was mainly accounted for by the amount in the RF, although there were some exceptions, particularly of low DD chemicals. While there was some variability due to cell outliers and donor variation, the overall reproducibility was very good. As six chemicals had to be applied in 100% ethanol due to low aqueous solubility, we compared the penetration of four chemicals with similar physicochemical properties applied in ethanol and phosphate-buffered saline. Of these, the DD of hydrocortisone was the same in both solvents, while the DD of propylparaben, geraniol and benzophenone was lower in ethanol. Some chemicals displayed an infinite dose kinetic profile; whereas, the cumulative absorption of others into the RF reflected the finite dosing profile, possibly due to chemical volatility, total absorption, chemical precipitation through vehicle evaporation or protein binding (or a combination of these). These investigations provide a substantial and consistent set of skin penetration data that can help improve the understanding of skin penetration, as well as improve the prediction capacity of in silico skin penetration models.

In vitro-to-in vivo extrapolation (IVIVE) by PBTK modeling for animal-free risk assessment approaches of potential endocrine-disrupting compounds.

While in vitro testing is used to identify hazards of chemicals, nominal in vitro assay concentrations may misrepresent potential in vivo effects and do not provide dose-response data which can be used for a risk assessment. We used reverse dosimetry to compare in vitro effect concentrations-to-in vivo doses causing toxic effects related to endocrine disruption. Ten compounds (acetaminophen, bisphenol A, caffeine, 17α-ethinylestradiol, fenarimol, flutamide, genistein, ketoconazole, methyltestosterone, and trenbolone) have been tested in the yeast estrogen screening (YES) or yeast androgen-screening (YAS) assays for estrogen and androgen receptor binding, as well as the H295R assay (OECD test guideline no. 456) for potential interaction with steroidogenesis. With the assumption of comparable concentration-response ratios of these effects in the applied in vitro systems and the in vivo environment, the lowest observed effect concentrations from these assays were extrapolated to oral doses (LOELs) by reverse dosimetry. For extrapolation, an eight-compartment Physiologically Based Toxicokinetic (PBTK) rat model based on in vitro and in silico input data was used. The predicted LOEL was then compared to the LOEL actually observed in corresponding in vivo studies (YES/YAS assay versus uterotrophic or Hershberger assay and steroidogenesis assay versus pubertal assay or generation studies). This evaluation resulted in 6 out of 10 compounds for which the predicted LOELs were in the same order of magnitude as the actual in vivo LOELs. For four compounds, the predicted LOELs differed by more than tenfold from the actual in vivo LOELs. In conclusion, these data demonstrate the applicability of reverse dosimetry using a simple PBTK model to serve in vitro-in silico-based risk assessment, but also identified cases and test substance were the applied methods are insufficient.

Uterine adenocarcinoma in the rat induced by afidopyropen. An analysis of the lesion's induction, progression and its relevance to humans.

Afidopyropen is a novel insecticide that acts as a TRPV channel modulator in chordotonal organs of target insects. In two carcinogenicity studies with Fischer rats, an increased incidence of uterine adenocarcinomas was observed at 1000 and 3000 ppm. This finding prompted an investigation of the mechanism of the tumor formation as well as the relevance of this mechanism to humans. The mechanistic work took parallel paths: one path investigated the pharmacokinetic properties of the test substance at the doses where the tumors were found; while the second path examined the key mechanistic events that culminated in uterine adenocarcinomas. The results of the investigation indicated that the tumors only occurred at doses where excretion of test substance was saturated - indicating that homeostatic biological and/or physiological processes were overwhelmed. At the doses where these processes were overwhelmed, the test substance acted through a mechanism of dopamine agonism, triggering a cascade key events that resulted in uterine adenocarcinomas. An analysis of these mechanisms observed in rat showed that they are both quantitatively (pharmacokinetic mechanism) and qualitatively (dopamine agonism mechanism) not relevant to humans. Therefore the uterine adenocarcinomas observed in the rat associated with high doses of Afidopyropen are not expected to pose a carcinogenic risk to humans.

Towards a generic physiologically based kinetic model to predict in vivo uterotrophic responses in rats by reverse dosimetry of in vitro estrogenicity data.

Physiologically based kinetic (PBK) modelling-based reverse dosimetry is a promising tool for the prediction of in vivo developmental toxicity using in vitro concentration-response data. In the present study, the potential of this approach to predict the dose-dependent increase of uterus weight in rats upon exposure to estrogenic chemicals was assessed. In vitro concentration-response data of 17ß-estradiol (E2) and bisphenol A (BPA) obtained in the MCF-7/BOS proliferation assay, the U2OS ER-CALUX assay and the yeast estrogen screen (YES) assay, were translated into in vivo dose-response data in rat, using a PBK model with a minimum number of in vitro and in silico determined parameter values. To evaluate the predictions made, benchmark dose (BMD) analysis was performed on the predicted dose-response data and the obtained BMDL10 values were compared with BMDL10 values derived from data on the effects of E2 and BPA in the uterotrophic assay reported in the literature. The results show that predicted dose-response data of E2 and BPA matched with the data from in vivo studies when predictions were made based on YES assay data. The YES assay-based predictions of the BMDL10 values differed 3.9-fold (E2) and 4.7- to 13.4-fold (BPA) from the BMDL10 values obtained from the in vivo data. The present study provides the proof-of-principle that PBK modelling-based reverse dosimetry of YES assay data using a minimum PBK model can predict dose-dependent in vivo uterus growth caused by estrogenic chemicals. In future studies, the approach should be extended to include other estrogens.

Dermal absorption for pesticide health risk assessment: Harmonization of study design and data reporting for North American Regulatory submissions.

Although an internationally-adopted in vitro dermal absorption test guideline is available (OECD Test Guideline 428), the replacement of the in vivo approach in North America for pesticide formulations has not occurred due to concern over the reliability and consistency of the in vitro results. A 2012 workshop convened a panel of experts in the conduct of in vitro studies used for pesticide risk assessment, together with North American regulators, to examine techniques for in vitro dermal absorption testing. Discussions led to the recommended "best practices" for the conduct of in vitro dermal absorption studies provided herein. The workshop participants also developed recommendations for reporting study results in order to improve the quality and consistency of the data submitted to regulatory agencies in North America. Finally, a case study is presented that illustrates the use of the "triple-pack" approach; the studies, conducted for the registration of sulfoxaflor, follow the standardized recommendations provided at the workshop. In addressing the concerns of these regulators and of the regulated community, and providing harmonized recommendations to facilitate comparative data analyses, it is anticipated that wider acceptance of in vitro dermal absorption studies alone can be achieved for pesticide risk assessment.

Intrinsic Xenobiotic Metabolizing Enzyme Activities in Early Life Stages of Zebrafish (Danio rerio).

Early life stages of zebrafish (Danio rerio, zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology.

A protocol to determine dermal absorption of xenobiotica through human skin in vitro.

Determination of the absorption through the skin is of utmost importance for predictions of benefits and of risks of dermal exposure to xenobiotica. In order to allow for flexibility, the OECD guideline for the determination of skin absorption for different purposes and use conditions (OECD guideline 428 combined with the Technical Guidance Document 28) is inexplicit; hence, different experimental procedures are used which may lead to limited comparability of study results. The here described protocol provides explicit guidance, whereas it does not invalidate other procedures within the frame of the OECD guideline since uncritical versus critical steps are differentiated. Optimizations are presented which finally led to a precisely defined protocol allowing for enhanced comparability of future study results. Some salient properties of this protocol are the storage of the prepared diffusion cell overnight refrigerated in the presence of a protease inhibitor cocktail and include investigation of the integrity of the skin sample as well as the removal of the upper stratum corneum by tape strips under standardized conditions.